Shape dependency of gold nanorods through TMB2+-mediated etching for the visual detection of NT-proBNP

Heart failure (HF) is a major public health problem triggered by heart circulation disorders. Early detection and diagnosis are conducive to the prevention and treatment of HF. Hence, it is necessary to establish a simple and sensitive method to monitor the diagnostic biomarkers of HF. The N-terminal B-type natriuretic peptide precursor (NT-proBNP) is acknowledged as a sensitive biomarker. In this study, a visual detection method for NT-proBNP was developed based on the oxidized 3,3′,5,5′-tetramethylbenzidine (TMB2+)-mediated etching of gold nanorods (AuNRs) and double-antibody-sandwich ELISA. The etching color for different amounts of NT-proBNP was obvious and significant differences could be ascertained based on the blue-shift of the longitudinal localized surface plasmon resonance (LLSPR) of the AuNRs. The results could be observed by the naked eye. The constructed system showed a concentration range from 6 to 100 ng mL−1 and a low detection limit of 6 ng mL−1. This method exhibited negligible cross-reactivity toward other proteins, and the recoveries of the samples ranged from 79.99% to 88.99%. These results demonstrated that the established method is suitable for the simple and convenient detection of NT-proBNP.


Introduction
Heart failure (HF) is a condition that affects 26 million individuals globally and has recently emerged as the leading cause of cardiovascular disease-related mortality and morbidity. 1 An aging population will result in a major rise in the prevalence of HF and associated healthcare costs. N-terminal pro-B-type natriuretic peptide (NT-proBNP) is currently regarded as the gold standard for the diagnosis and prognosis of HF. [2][3][4][5] Therefore, it is crucial to create a sensitive, specic, and precise NT-proBNP detection technique.
Traditional immunoassays as well as contemporary biosensors are two of the analysis techniques that have been developed and used to detect NT-proBNP. The usage of earlier techniques, such as enzyme-linked immunosorbent assay (ELISA) and immunochromatographic test, is constrained by the need for basic equipment, like microplate readers and strip readers. [6][7][8][9][10][11][12] The latter assays include electrochemical biosensors, photoelectrochemical biosensors, surface plasma resonance biosensors, electrochemiluminescence biosensors, and micro-uidic immunoassay biosensors, among others. [13][14][15][16][17][18][19][20][21][22] Large instruments are necessary for these delicate biosensors, which are expensive, uncomfortable for users, and need trained technicians. Due to these aws, it is a challenge to meet the demand for NT-proBNP monitoring in insufficient experimental settings and less developed countries.
Naked-eye colorimetric detection based on nanomaterials has recently drawn a lot of interest. For instance, assays of Ochratoxin A, E. coli O157, PSA, and HIV-1 capsid antigen p24 have been studied. [23][24][25] Gold nanoparticles (AuNPs) are good signal indicator, which can easily cause dramatic color change by slightly changing their shapes, sizes, compositions and aggregation states. The AuNPs solution obtained with an analyte target was found to produce a single color shi that was plainly visible in the reaction solution. As a result, it might be challenging to discriminate between uctuations in optical density (without a spectral peak shi) and to precisely measure the concentration of the target molecules by the naked eye. The ability of human eyes to detect subtle color differences is well recognized (with spectral peak shis). Gold nanorods (AuNRs) have been used as a colorful chromogenic substrate to detect a large number of target molecules because AuNRs display different multicolored and variable longitudinal plasmon wavelengths. 26 The morphology of AuNRs obtained through oxidized 3,3 ′ ,5,5 ′ -tetramethylbenzidine (TMB 2+ ) generated by HRP and E-mail: hy0211085@hainmc.edu.cn; phzmh61@aliyun.com; wuqiang001001@aliyun.com the idea of antigen-antibody combination were used in this demonstration to showcase a portable and visible detection method for quantifying NT-proBNP.

Instruments
The images of the surface morphology of the gold nanorods (AuNRs) were obtained using an Hitachi HT 7800 transmission electron microscopy system (TEM) (Hitachi, Japan). An incubator was purchased from Shanghai Bosun Industrial Co., Ltd. The constant temperature water bath was provided by Shanghai Yiheng Scientic Instrument Co., Ltd. The blue-shis of the longitudinal localized surface plasmon resonance (LLSPR) of the AuNRs were measured using a Bio-Tek Synergy HTX microplate reader (Winooski, USA), and using a Vortex Mixer purchased from Eppendorf Company (Hamburg, Germany).

Synthesis of AuNRs
AuNRs were prepared according to the literature with several modications. 27 Briey, (1) the seed liquid was prepared as follows: 2.5 mL of 0.2 M cetyltrimethylammonium bromide (CTAB) was transferred into a 10 mL conical ask and frequently stirred at 400 rpm min −1 with a magnetic stirrer. Then, 2.5 mL of 0.5 mM HAuCl 4 solution was added. Subsequently, 0.3 mL of 0.01 M freshly prepared NaBH 4 solution was added. The mixed liquid was incubated at 30°C for 30 min; (2) the growth solution was prepared as follows: 9 g of CTAB and 250 mL of ultrapure water were added into a conical ask, with continuous heating and stirring until the CTAB was completely dissolved, and aerward, 1.1 g of 5-bromosalicylic acid was added. Aer stirring for 5 min, 12 mL of 4 mM AgNO 3 solution was added to the solution and incubated at 30°C for 15 min. Then, 250 mL of 1 mM HAuCl 4 solution was added, and the solution gradually turned orange-red. Subsequently, 2 mL of 0.064 M ascorbic acid (AA) was dropped into the conical ask.
The color of the mixed solution rapidly changed from orangered to colorless. Aerward, 800 mL of seed liquid was added and mixed for 30 s. Finally, the conical ask was le undisturbed at 30°C for 24 h. Aer that, the solution was centrifuged at 8000 g min −1 for 15 min, and the supernatant was removed. The AuNRs precipitate was resuspended with puried water and placed in a 30°C incubator for further use.

HRP-labeled Ab2 (HRP-Ab2)
The HRP was labeled with Ab2 according to the kit instructions for activating HRP reagents. Briey, 25 mL of Ab2 (4 mg mL −1 ) was transferred into a 1.5 mL tube. Then, 100 mL reagent I activated HRP (10 mg mL −1 ) was added. The pH of the mixture solution was adjusted to 9.5 with reagent II (about 20 mL) and then reacted for 1 h at 37°C. Aerward, reagent III was dropped into the tube and stored at 4°C for further use.
Procedures for the visual detection of NT-proBNP Ab1 in pH 9.6 carbonate bicarbonate buffer was added into 96well plates for incubation at 37°C. Then, each well was washed three times with pH 7.2 PBS containing 0.05% tween 20 (PBST). Subsequently, different concentrations of NT-proBNP (in PBS buffer containing 5% BSA) were added to the plates and incubated at 37°C. Aer three times washing, each well was dispensed with 100 mL HRP-Ab2 and incubated at 37°C. Aer further washing, TMB substrate solution was pipetted into each well and reacted at 37°C. Then, the reaction was stopped by adding 2 M H 2 SO 4 . Aer that, AuNRs solution was placed into each well and reacted at 37°C. Finally, photographs were taken by a smartphone and the corresponding absorbance at 500-900 nm was measured using a Bio-Tek Synergy HTX microplate reader.

Optimization of the experimental conditions
To obtain high sensitivity, several experimental parameters of the proposed NT-proBNP assay were optimized by altering the following: coating time from 30 min to 120 min; incubation time for NT-proBNP from 30 min to 120 min; incubation time for HRP-Ab2 from 30 min to 120 min; and etching time for AuNRs from 5 min to 20 min.

Establishment of the NT-proBNP standard colorimetric card
The standard multicolor card was established by adding different concentrations of NT-proBNP according to the procedures for the visual detection of NT-proBNP under the above optimization conditions, except the concentration of NT-proBNP added ranged from 3 to 100 ng mL −1 . Meanwhile, the LLSPR peak shi of the AuNRs was recorded using a Bio-Tek Synergy HTX microplate reader.

Method specicity
To demonstrate the specicity of the purposed method, serum amyloid A antigen (SAA), C-reactive protein (CRP), heparinbinding protein antigen (HBP), P1 protein of mycoplasma pneumoniae (P1), and P30 protein of mycoplasma pneumoniae (P30) with a concentration of 20 ng mL −1 were tested. The operation steps were the same as in the detection procedure for the establishment of the NT-proBNP standard colorimetric card.

Detection of NT-proBNP in the serum samples
To verify the accuracy and feasibility of the established method for serum samples, three different concentrations of NT-proBNP were spiked in 10-fold diluted healthy human serum without NT-proBNP. The experimental steps were the same as those for the establishment of the NT-proBNP standard colorimetric card.

Characterization of AuNRs
As shown in Fig. 1, AuNRs were created using a seed-mediated approach and were identied by their TEM images and UV-vis absorption. The TEM picture of the produced AuNRs revealed good consistency. The typical longitudinal and transverse bands were found in the UV-vis absorption spectrum at wavelengths of 520 nm and 730 nm, respectively. These ndings show that the AuNRs were properly prepared.

Principle behind the established visual detection of NT-proBNP
The principle of visually detecting NT-proBNP was illustrated in Fig. 2. A sandwich ELISA assay was employed in our investigation. HRP-Ab2 was immobilized on the wells aer the formation of the double-antibody-sandwich immune complex. The amount of HRP immobilized on the wells was positively proportional to the concentration of NT-proBNP in the sample. Then, TMB 2+ (oxidized TMB) was produced by TMB in the presence of HRP. Finally, AuNRs solution was added in and shortened by the etching effect of TMB 2+ in the presence of CTAB, which caused a blue-shi of the longitudinal localized surface plasmon resonance (LLSPR) peak, while the transverse localized surface plasmon resonance (TLSPR) peaks were nearly unaffected. Fig. 1 shows the TEM images and UV-vis spectra of the prepared AuNRs. The LSPR and TSPR of the AuNRs were located at 730 nm and 520 nm, respectively. The length of the AuNRs was about 60 nm. With the addition of various concentrations of NT-proBNP, the length of the AuNRs was shortened by various degrees. The TEM images are shown in Fig. 3. These results demonstrate the proposed method is feasible and can be used for the quantitative detection of NT-proBNP.

Optimization of the conditions of the immunosensor
The incubation times of Ab1, NT-proBNP, and HRP-Ab2 were all tuned in order to achieve the best sensitivity. The optimizated results are shown in Fig. 3A-D. The incubation times for Ab1, NT-proBNP, HRP-Ab2, and AuNRs were 120, 120, 60, and 20 min, respectively, according to the creation of a striking blue-shi (Fig. 4).

Calibration curve for NT-proBNP detection
With the rise in NT-proBNP concentration from 6 to 100 ng mL −1 , an extraordinary color change from brown to blue and ultimately   to yellow was clearly visible to the unaided eye, as shown in Fig. 5A. The color of the well of 3 ng mL −1 was just like the color of the negative well, which was brown. Meanwhile, the LLSPR peak gradually shied from 730 nm to 520 nm with the increase in NT-proBNP concentration, as shown in Fig. 5B. The calibration plots show the good linear relationship between the blue-shis of the LLSPR peak (Dl max ) and NT-proBNP concentration in the range from 6 to 50 ng mL −1 , y = 4.1036x − 4.3271 (R 2 = 0.99). Hence, the visual assay established could be applied to evaluate NT-proBNP concentrations in decient experimental conditions.

Specicity of the established immunosensor
Several analogs were found using targeted investigations to gauge the immunosensor's specicity. In the presence of 20 ng mL −1 of NT-proBNP, SAA, CRP, HBP, P1, and P30, there was a peak shi of the LLSPR, as shown in Fig. 6. While the other proteins exhibited negligible peak-shis, only 20 ng mL −1 of NT-proBNP clearly blue-shied the LSPR of AuNRs. Because of this, the suggested immunosensor showed high selectivity for NT-proBNP.

Recovery test of NT-proBNP in serum samples
To assess the effectiveness of the analytical process serum samples were spiked to 12, 25, and 50 ng mL −1 . The ndings in Table 1 show that the NT-proBNP recoveries fell between the ranges of 79.99% and 88.99%, indicating that the established optical detection for NT-proBNP had a high degree of accuracy.

Conclusions
The conventional ELISA for detecting NT-proBNP, which has strong repeatability and specicity, was proposed to be more convenient in this study using a novel chromatic method based on AuNRs. The assay could be successfully recovered aer being used to spike clinical samples. Unlike the conventional ELISA, this immunoassay can be applied in the absence of ideal experimental circumstances and can be visually identied without the use of specic equipment. Furthermore, by simply    changing the relevant antigen and antibody, this approach can be quickly applied to other diagnoses.

Conflicts of interest
The authors declare that they have no competing interests.